This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. My project utilized living, adult Drosophila to investigate the mechanisms involved in olfactory associative learning. Drosophila can be taught to associate an odor with an aversive stimulus, (=electric shock), and subsequently avoid that same odor. Mutants expressing a fluorescent calcium reporter, (camgaroo), in brain areas, (the Mushroom Bodies), known to be involved in this form of learning were used. We have been able to apply precise, known quantities of the neurotransmitters thought to be involved directly to Mushroom Body neurons, (Kenyon cells). We have observed increases in intracellular calcium with acetylcholine, (mimicing the odor input), dopamine, (electric shock- aversive stimulus), and an interesting modulation of the standard acetylcholine response when dopamine is co-applied as would occur during learning. We are the first people to directly investigate the function of these neurons in a living, adult fly and obtain quantitative data. In addition, as requested by reviewers, we have also obtained peliminary data indicating the nature of the dopamine receptor involved and observed modulation of the acetylcholine response with the important secondary messenger cyclic adenosine monophosphate: this molecule has been heavily implicated in this type of learning but only indirectly until now. This work is an important step in the determination of the mechanisms involved in this form of learning and the first at this level in the intact fly.